Analytical separation of tea catechins and food-related polyphenols by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) using the type-J coil planet centrifuge was applied to compositional analysis of tea catechins and separation of other food-related polyphenols. The HSCCC separation of nine different standard compounds and those from extracts of commercial tea leaves was performed with a two-phase solvent system composed of tert-butyl methyl ether-acetonitrile-0.1% aqueous trifluoroacetic acid (TFA) (2:2:3, v/v/v) by eluting the upper organic phase at a flow rate of 2 ml/min. The main compounds in the extract of non-fermented green tea were found to be monomeric catechins, their galloylated esters and caffeine. In addition to these compounds, oxidized pigments, such as hydrophobic theaflavins (TFs) and polar thearubigins (TRs) were also separated and detected from the extracts of semi-fermented oolong tea and fermented black tea. Furthermore, several food-related polyphenols, such as condensed catechin oligomers (procyanidins), phenolic acids and flavonol glycosides were clearly separated under the same HSCCC condition. These separation profiles of HSCCC provide useful information about the hydrophobic diversity of these bioactive polyphenols present in various types of teas and food products.     

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples

The potential and applicability of UHPSFC–MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC–MS/MS and UHPSFC–MS/MS in both ESI+ and ESI− modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7 min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC–MS/MS and for 32% in UHPSFC–MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC–MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.     

Identification/quantification of multiple pesticide residues in food plants by ultra-high-performance liquid chromatography-time-of-flight mass spectrometry

In this study, the potential of ultra-high-performance liquid chromatography coupled with the time-of-flight mass spectrometry (UHPLC–TOF MS) to enable rapid and comprehensive analysis of 212 pesticide residues in QuEChERS extracts obtained from four plant matrices has been investigated. Method optimization is discussed in detail. In addition to molecular adducts, also fragment ions were provided for all target pesticides, thus obtaining at least three identification points required by European Decision 2002/657/EC was achieved. To get maximum information on analytes present in the extracts, each sample was examined within two injections, the first in a positive and the next one in a negative ionization mode. Under UHPLC conditions, both analyses were completed within 24 min. For more than 96% of pesticides involved in this study, the limit of quantification was ≤10 μg/kg. As a part of the work, strategy enabling screening of non-target pesticides and their metabolites is demonstrated on analysis of real-life samples.     

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 μM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.     

超高效液相色谱-高分辨质谱联用结合整合过滤策略全面分析茶树花中化学成分

茶树花与茶鲜叶同为茶树的生物产出,但茶树花往往被视为茶叶生产过程中的废物被舍弃,造成了茶树花资源的极大浪费.目前对于茶树花中化学成分的分析主要集中在氨基酸、茶多酚等单一类型化学成分上,对于茶树花中多类化学成分的同时分析仍鲜见报道.研究者对于茶树花中所含化学成分的种类和含量不完全清楚,成为制约茶树花深度开发与利用的重要原...     

Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition

Tea is rich in polyphenols and has a variety of biological activities. In order to better understand the biological effects of tea constituents on human health, markers for their exposure and their metabolic fates are needed. Previously, we have characterized several catechin metabolites in the blood and urine, but more information on the metabolite profile of tea polyphenols is needed. In the present study, the human urinary metabolite profile of tea polyphenols was investigated using liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. With data-dependent MS/MS analysis by collecting the MS2 and MS3 spectra of the most intense ions in the sample, we identified more than twenty metabolites of tea polyphenols from human urine samples. (-)-Epigallocatechin (EGC) glucuronide, methylated EGC glucuronide, methylated EGC sulfate, (-)-epicatechin (EC) glucruronide, EC sulfate, methylated EC sulfate, as well as the glucuronide and sulfate metabolites of the ring-fission metabolites of tea catechins, 5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4), 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M6'), were the major human urinary metabolites of tea polyphenols. To our knowledge, this is the first report of the direct simultaneous analysis of the human urinary metabolite profile of tea polyphenols using single sample analysis. This method can also be used for thorough investigations of the metabolite profiles of many other dietary constituents.     

Determination of eight penicillin antibiotics in pharmaceuticals,milk and porcine tissues by nano-liquid chromatography

This study describes the ability of nanoscale liquid chromatography (nano-LC) coupled with UV or mass spectrometry (MS) for the simultaneous determination of eight common penicillin antibiotics (amoxicillin, ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, nafcillin and dicloxacillin) in commercial samples (pharmaceuticals, milk, porcine tissues (liver and kidney)) for the first time. Material types of the on-column polymeric frits (polystyrene-based and polymethacrylate-based monoliths) and the packed stationary phase materials (C8 and C18 particles of 3 μm) used in the nano-LC for the influence of penicillin separation were evaluated. The nano-LC and MS parameters such as the composition and flow rate of mobile phase, capillary voltage and temperature of dry gas were examined in order to acquire high separation resolution and detection sensitivity for penicillin analyses. Furthermore, a home-made in-line filter (a nylon membrane of 0.2 μm pore size), was first used to connect with the flow cell of high sensitivity UV detector or the nanoelectrospray needle in MS detection. The result indicated it could effectively improve the reproducibility of penicillin mass signals or prolong the lifetime of the flow cell. The nano-LC methods provided good quantitative precisions in the range of 89.5–111.2% for UV detection at 0.5 μg/mL penicillins, and 83. 1–94.9% for MS detection at 5 μg/L penicillins), respectively, as well as offered stable retention repeatabilities (the relative standard deviation (RSD) of retention time was lower 0.30% in both the UV and MS detections). Compared to other LC–MS methods, the proposed nano-LC systems provided better detection sensitivity for these penicillins (the limits of detection (LOD) was of 2.27–4.06 μg/L for UV mode, and 0.01–0.51 μg/L for MS mode) when either UV or MS detector was employed.     

Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins,three purine alkaloids,and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C₁₈ reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.     

Rapid qualitative and quantitative ultra high performance liquid chromatography method for simultaneous analysis of twenty nine common phenolic compounds of various structures

Twenty nine phenolic compounds comprising nine phenolic acids, sixteen flavonoids (including eight tea catechins, glycosides and aglycones), four coumarins plus caffeine were analysed within 20 min using ultra high performance liquid chromatography (UHPLC) with PDA detection. UHPLC system was equipped with C18 analytical column (100 mm × 2.1 mm, 1.7 μm), utilising 0.1% formic acid and methanol mobile phase in the gradient elution mode. The developed method was tested for the system suitability: resolution, asymmetry factor, peak capacity, retention time repeatability and peak area repeatability. The method was fully validated in the terms of linearity (r² > 0.9990 for all 30 compounds), range (typically 1-100 mg L⁻¹), LOD, LOQ, inter/intra-day precision (<3% and <9% respectively) and inter/intra-day accuracy (typically 100 ± 10%). Subsequently the method was applied to the identification (spectral information and peak purity calculations were profited) and quantification of phenolic compounds and caffeine present in tea infusions and extracts.     

Rapid high performance liquid chromatography method development with high prediction accuracy,using 5 cm long narrow bore columns packed with sub-2 μm particles and Design Space computer modeling

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5 cm × 2.1 mm columns packed with sub-2 μm particles and computer simulation (DryLab^® package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3–5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p > 400 bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.     

Determination of naphthalene-derived compounds in apples by ultra-high performance liquid chromatography-tandem mass spectrometry

Naphthylacetic acid, naphthyloxy acetic acid and naphthylacetamide belong to a group of synthetic substances known as “auxin-like” compounds which are used as growth regulators in vegetables and fruits due to their structure similarities with the indoleacetic acid, the most important plant auxin. This paper reports a selective, sensitive and fast ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for the determination of naphthylacetamide (NAD) and the isomers (α and β) of naphthylacetic acid (NAA) and naphthyloxy acetic (NOA) acid in apple samples. A baseline separation between the respective isomers was achieved using an RP-Amide column with gradient elution. The UHPLC-MS/MS method developed, using electrospray and selected reaction monitoring (SRM) acquisition mode led to a reliable determination of these family of compounds in apple samples at low quantitation levels, down to 1.0 μg kg⁻¹ and 0.25 μg kg⁻¹ respectively. For confirmation of NAA accurate mass measurement is proposed giving at these conditions quantitation limits of 10 μg kg⁻¹ for this compound. The UHPLC-MS/MS method developed was used for the analysis of apple samples harvested in three different apple fields from Lleida (Spain) during the blooming period. NAD and NAA were found in samples collected during 4–5 weeks after application at concentrations between the quantification limits and 43 μg kg⁻¹ and 24 μg kg⁻¹, respectively.     

Application of amino‐based chitosan cyclodextrin derivatives for the extraction of catechins in green tea with high‐performance liquid chromatography

Chitosan derivatives have been studied widely, but poor solubility in water restricts their applications. In this study, four types of amine‐based chitosan derivatives were prepared and modified further with beta‐cyclodextrin. The sequential microextraction of catechins ((+)‐catechin and (?)‐epigallocatechin gallate) from green tea powder by an optimized solid‐phase extraction method using these four derivatives was investigated. The optimal conditions for the extraction of catechins were 60°C for a 40 min extraction period. The purity and amount of each catechin were determined by high‐performance liquid chromatography. The different amines strengthened the extraction capacity of chitosan. Among the four types of amines, ethylene diamine grafted chitosan beta‐cyclodextrin had the highest extraction capacity to catechins. Therefore, this material was used in the extraction assay, and the standard curves of (+)‐catechin and (?)‐epigallocatechin gallate were linear over the concentration range, 0.25–500 µg/mL, after assaying five data points in duplicate. Solid‐phase extraction with the amino‐based chitosan beta‐cyclodextrin system is a new application of chitosan, which has potential applications in the extraction of bioactive compounds from plant materials or the removal of different impurities from specific extracts.     

Simple and rapid UV spectrophotometry of caffeine in tea coupled with sample pre-treatment using a cartridge column filled with polyvinylpolypyrrolidone (PVPP)

We have applied a sample pre-treatment method with a cartridge column filled with polyvinylpolypyrrolidone (PVPP) to the effective removal of polyphenols and simple UV spectrophotometry of caffeine in tea. The absorption maximum length (lambda(max)) for caffeine was close to those for tea catechins in aqueous 1% acetic acid; therefore, the UV spectrum of a non-treated green tea sample had a large absorption wave. In contrast, the absorbance of the green tea sample was gradually reduced by PVPP cartridge treatment using PVPP from 0 to 50 mg, and was nearly constant using a pre-treatment cartridge with more than 100 mg PVPP, because tea catechins were effectively removed and caffeine was mostly recovered from a green tea sample by means of PVPP cartridge treatment. The PVPP pre-treatment cartridge also removed polyphenols successfully from oolong and black tea samples. Comparison with conventional HPLC analysis indicated that the present pre-treatment method with a PVPP cartridge was useful for the simple and selective UV spectrophotometric determination of caffeine in green, oolong and black tea samples.     

Feasibility of ultra-high performance liquid and gas chromatography coupled to mass spectrometry for accurate determination of primary and secondary phthalate metabolites in urine samples

Phthalates (PAEs) are ubiquitous toxic chemical compounds. During the last few years, some phthalate metabolites (MPAEs) have been proposed as appropriate biomarkers in human urine samples to determine PAE human intake and exposure. So, it is necessary to have fast, easy, robust and validated analytical methods to determine selected MPAEs in urine human samples. Two different instrumental methods based on gas (GC) and ultra-high performance liquid (UHPLC) chromatography coupled to mass spectrometry (MS) have been optimized, characterized and validated for the simultaneous determination of nine primary and secondary phthalate metabolites in urine samples. Both instrumental methods have similar sensitivity (detection limits ranged from 0.03 to 8.89 pg μL⁻¹ and from 0.06 to 0.49 pg μL⁻¹ in GC–MS and UHPLC–MS², respectively), precision (repeatability, expressed as relative standard deviation, which was lower than 8.4% in both systems, except for 5OH-MEHP in the case of GC–MS) and accuracy. But some advantages of the UHPLC–MS² method, such as more selectivity and lower time in the chromatographic runs (6.8 min vs. 28.5 min), have caused the UHPLC–MS² method to be chosen to analyze the twenty one human urine samples from the general Spanish population. Regarding these samples, MEP showed the highest median concentration (68.6 μg L⁻¹), followed by MiBP (23.3 μg L⁻¹), 5cx-MEPP (22.5 μg L⁻¹) and MBP (19.3 μg L⁻¹). MMP (6.99 μg L⁻¹), 5oxo-MEHP (6.15 μg L⁻¹), 5OH-MEHP (5.30 μg L⁻¹) and MEHP (4.40 μg L⁻¹) showed intermediate levels. Finally, the lowest levels were found for MBzP (2.55 μg L⁻¹). These data are within the same order of magnitude as those found in other similar populations.     

Photodiode array to charged aerosol detector response ratio enables comprehensive quantitative monitoring of basic drugs in blood by ultra-high performance liquid chromatography

Quantitative screening for a broad range of drugs in blood is regularly required to assess drug abuse and poisoning within analytical toxicology. Mass spectrometry-based procedures suffer from the large amount of work required to maintain quantitative calibration in extensive multi-compound methods. In this study, a quantitative drug screening method for blood samples was developed based on ultra-high performance liquid chromatography with two consecutive detectors: a photodiode array detector and a corona charged aerosol detector (UHPLC–DAD–CAD). The 2.1 mm × 150 mm UHPLC column contained a high-strength silica C18 bonded phase material with a particle size of 1.8 μm, and the mobile phase consisted of methanol/0.1% trifluoroacetic acid in gradient mode. Identification was based on retention time, UV spectrum and the response ratio from the two detectors. Using historic calibration over a one-month period, the median precision (RSD) of retention times was 0.04% and the median accuracy (bias) of quantification 6.75%. The median precision of the detector response ratio over two orders of magnitude was 12%. The applicable linear ranges were generally 0.05–5 mg L⁻¹. The method was validated for 161 compounds, including antipsychotics, antidepressants, antihistamines, opioid analgesics, and adrenergic beta blocking drugs, among others. The main novelty of the method was the proven utility of the response ratio of DAD to CAD, which provided the additional identification efficiency required. Unlike with mass spectrometry, the high stability of identification and quantification allowed the use of facile historic calibration.     

等度反相高效液相色谱法测定茶多酚中的儿茶素和咖啡因

 介绍一种简便的等度反相高效液相色谱分析茶多酚中 5种儿茶素和咖啡因的快速方法。样品总的分析时间在 0 5h内。色谱柱为ResolveC18;流动相为水 体积分数为 85 %的磷酸水溶液 乙腈 N ,N 二甲基甲酰胺 (DMF)(体积比为 85 9∶1∶12 0∶2 0 ) ;柱温为 43℃ ;紫外检测波长为 2 80nm。研究了流动相中改良剂DMF与容量因子的关系。被测组分的含量与其峰面积有良好的线性关系 (r =0 .9992～ 0 .9999) ;加标回收率在 83.33%～ 10 4.42 %(RSD在 0 .74%～ 1.43% )。     

Coupling capillary electrochromatography with mass spectrometry by using a liquid-junction nano-spray interface

Capillary electrochromatography (CEC) was coupled with mass spectrometry (MS) for the separation of some selected pesticides and drug enantiomers. CEC separations were carried out in fused silica capillaries packed with either 5 μm RP₁₈ silica or 5 μm silica modified vancomycin particles. The capillary column was connected with the MS utilizing a laboratory-made liquid-junction interface equipped with a 50 μm I.D. capillary-tip positioned at a few mm from the orifice of the MS. The CEC–MS set-up was operated without external pressure assistance during the electrochromatographic run commonly used to avoid bubble formation. However a hydrostatic pressure of a few kPa was applied only to the liquid-junction interface to optimize the ion-spray due to the low I.D. of the capillary-tip. In order to optimize the CEC–MS method, several experimental parameters were studied, namely the inlet pressure, the hydrostatic pressure into the liquid-junction interface, the type of sheath-liquid and the mobile phase. The application of an inlet pressure influenced only analyte retention times that were shortened by increasing the pressure. On the contrary the hydrostatic pressure applied to the interface increased the flow rate into the tip also increasing the ion-signal recorded in the mass spectrometry. The ion-signal raised almost linearly by increasing the outlet pressure till 3.5 kPa and then decreased. The separation of the selected pesticides was not influenced at all changing the hydrostatic pressure on the interface. Some basic enantiomeric compounds of pharmaceutical interest were successfully separated by CEC achieving good resolution. They were detected by MS with limit of detection in a range of 0.24–0.60 μg/mL.     

Statistical mixture design selective extraction of compounds with antioxidant activity and total polyphenol content from Trichilia catigua

Statistical design mixtures of water, methanol, acetone and ethanol were used to extract material from Trichilia catigua (Meliaceae) barks to study the effects of different solvents and their mixtures on its yield, total polyphenol content and antioxidant activity. The experimental results and their response surface models showed that quaternary mixtures with approximately equal proportions of all four solvents provided the highest yields, total polyphenol contents and antioxidant activities of the crude extracts followed by ternary design mixtures. Principal component and hierarchical clustering analysis of the HPLC–DAD spectra of the chromatographic peaks of 1:1:1:1 water–methanol–acetone–ethanol mixture extracts indicate the presence of cinchonains, gallic acid derivatives, natural polyphenols, flavanoids, catechins, and epicatechins.     

Magnetically immobilized frits for the preparation of packed columns used in capillary electrochromatography

Fabrication of porous frits to retain stationary phases is a critical issue in column preparation for capillary electrochromatography (CEC). In this work, porous frits were prepared by applying an external magnetic field to magnetically responsive particles placed inside a fused-silica capillary. Three batches of uniform magnetite spheres with particle diameters of 0.3, 0.4, and 0.6 μm and saturation magnetization values of 73.03, 74.41, and 77.83 emu/g, respectively, were used as frit particles and octadecyl- and phenyl-bonded silica gels were packed successfully into frit-containing capillaries. The performance of the resulting magnetically immobilized frits and packed columns was evaluated. The electroosmotic mobilities in capillaries containing outlet frit only were found to be reduced by 2–4% whereas the plate heights of an unretained marker increased by 30–50% as compared to those in open capillaries. These variations are believed to be associated with the inhomogeneities of the packed structure of the frits. The magnetically immobilized frits showed adequate mechanical strength to withstand the flow drag force, allowing separation in capillaries packed with 5-μm stationary phases up to 10–15 cm, thus rendering column efficiency and reproducibility comparable with those obtained with sintered frits. Taken together, retaining frits made of uniform magnetite particles serves as a viable alternative to sintered frits for column preparation, which offers several distinct advantages such as ease of preparation, improved durability as compared to sintered frits where the removal of the polyimide coating makes the packed column susceptible to breakage, and use of large-bore capillaries for semipreparative separations.     

Constituents of the green tea seeds of Camellia sinensis

Green tea (Camellia sinensis) leaves are known to contain active ingredients such as catechins and caffeine, and are widely useful materials. Recently, green tea flowers also have been in the spotlight. However, little attention has been paid to the tea seeds. In this work, the constituents of green tea seeds and green tea leaves were compared. Caffeine was found in the seeds, whereas catechins (usually obtained from green tea leaves) were not observed. Next, we investigated the constituents of hexane extracts and methanol extracts of green tea seeds. We found that the hexane extracts contained high amounts of oleic glyceride (79.9%) in addition to linoleic glyceride (20%). We confirmed the structures of these glycerides by NMR spectroscopy and by synthesis from a fatty acid and glycerol. The methanol extract was found to contain naringenin glucosides by mass spectrometry and NMR spectroscopic analysis.